Composition of matter and process

ABSTRACT

Novel antibiotic U-64,815 producible in a fermentation under controlled conditions using a biologically pure culture of the microorganism Streptomyces microspinus, NRRL 12524. This antibiotic is active against various Gram-positive bacteria, for example, Staphylococcus aureus. It is also active against Haemophilus influenzae. Thus, antibiotic U-64,815 can be used in various environments to eradicate or control such bacteria.

BACKGROUND OF THE INVENTION

Antibiotic U-64,815 can be differentiated from the known relatedantibiotics kijanimicin [A. K. Mallams, et al., J. Amer. Chem. Soc. 103,3938, 3940 (1981)], antlermicins [K. Kobinata, et al., J. Antibiotics33, 244 (1980); K. Isono, et al., ibid., 33, 772 (1980)] andtetrocarcins [F. Tomita, et al., J. Antibiotics 33, 668 (1980); T.Tamaoki, et al., ibid., 33, 946 (1980)] on the basis of observed ¹³ Cmagnetic resonance spectra. Compound 12 disclosed in the J.A.C.S.article, appears to be identical to antibiotic U-64,815. Finalresolution of this identity is in progress.

BRIEF SUMMARY OF THE INVENTION

Antibiotic U-64,815 is producible in a fermentation under controlledconditions using a biologically pure culture of the new microorganismStreptomyces microspinus, NRRL 12524.

Antibiotic U-64,815 has moderate to excellent in vitro activity againstall strains of Haemophilus influenzae tested. It is also active againstvarious Gram-positive bacteria. Further, the base addition salts ofantibiotic U-64,815 are also active against these bacteria. Thus,antibiotic U-64,815 and its salts can be used to disinfect washed andstacked food utensils contaminated with S. aureus. They can also be usedas disinfectants on various dental and medical equipment contaminatedwith S. aureus. Still further, antibiotic U-64,815 and its salts can beused as a bacteriostatic rinse for laundered clothes, and forimpregnating papers and fabrics; and, they are also useful forsuppressing the growth of sensitive organisms in plate assays and othermicrobiological media.

DETAILED DESCRIPTION OF THE INVENTION

Chemical and Physical Properties of Antibiotic U-64,815:

Molecular Weight Range: 1,104-1,324 (based on molecular formula).

Molecular Formula: C₆₀₋₆₅ H₈₂₋₁₀₂ N₁₋₃ O₁₈₋₂₅.

Ultraviolet Absorption Spectrum:

The UV spectrum of antibiotic U-64,815 is shown in FIG. 2 of thedrawings. The solution of antibiotic U-64,815 in ethanol displayedabsorption as follows:

    ______________________________________                                               Solvent       λ max                                             ______________________________________                                               Ethanol       215                                                                           240                                                                           267                                                                           278 (sh)                                                        .1 N HCl      213                                                             in Ethanol    261                                                      ______________________________________                                    

Melting Point: 185-187 (dec)

Infrared Absorption Spectrum:

Antibiotic U-64,815 has a characteristic infrared absorption spectrum ina mineral oil mull as shown in FIG. 1 of the drawings. Peaks areobserved at the following wave lengths:

    ______________________________________                                        Band                      Band                                                Freq.  Inten.  Type       Freq.   Inten.                                                                              Type                                  ______________________________________                                        3480.0 40      SH       *   1196.9  43    BRD                                 3442.3 40      BRD          1170.9  18    AVG                                 3280.0 56      SH       *   1149.7  18    SH                                  3058.5 67      SH           1141.9  17    AVG                                 3025.7 56      AVG          1127.5  10    AVG                                 2956.2 0       BRD      M   1095.6  8     AVG                                 2931.1 0       BRD      M   1059.0  6     AVG                                 2870.4 3       BRD      M   1014.6  10    AVG                                 2854.0 2       AVG      M   1000.2  14    AVG                                 2726.7 70      BRD      M   981.8   14    AVG                                 2661.1 71      AVG          936.5   51    SH                                  2617.7 76      SH           927.8   43    SHP                                 1760.2 16      AVG          914.3   56    AVG                                 1729.3 19      AVG          897.0   56    SHP                                 1629.0 31      AVG          865.1   37    AVG                                 1575.0 37      SH       *   831.4   55    AVG                                 1544.2 10      AVG          800.5   70    AVG                                 1510.4 43      AVG          779.3   55    AVG                                 1455.4 9       AVG      M   730.1   51    AVG                                 1413.9 37      AVG          704.1   62    AVG                                 1378.3 12      AVG      M   667.4   57    SH                                  1366.7 20      AVG          656.8   54    AVG                                 1346.4 26      AVG          619.2   54    SH                                  1311.7 25      AVG          607.6   50    AVG                                 1250.9 26      AVG                                                            1220.1 31      AVG                                                            ______________________________________                                         Band Freq.: Band Frequencies in Wavenumbers (CM.sup.-1)                       Inten.: Intensity in Percent Transmittance (% T)                              Date Type in Local Peak Region:                                               BRD = Broad;                                                                  AVG = Average;                                                                SHP = Sharp;                                                                  SH = Shoulder                                                                 Peak List Edited.                                                             * Indicates Peaks Added.                                                      M: Possible Interference from Mineral Oil.                               

Antibiotic U-64,815 has a characteristic infrared absorption spectrumwhen passed into a KBr pellet. Peaks are observed at the following wavelengths:

    ______________________________________                                        Band                     Band                                                 Freq.  Inten.  Type      Freq.   Inten.                                                                              Type                                   ______________________________________                                        3480.0 32      SH      *   1170.9  27    AVG                                  3441.4 30      BRD         1141.0  26    AVG                                  3259.1 52      SH          1126.5  18    AVG                                  3029.5 55      AVG         1096.6  14    AVG                                  2971.6 31      AVG         1058.0  11    AVG                                  2931.1 16      AVG         1014.6  16    AVG                                  2911.8 24      SH          999.2   20    AVG                                  2852.0 36      SHP         981.8   21    AVG                                  2799.0 66      SH          937.5   55    AVG                                  2662.0 67      AVG         927.8   50    AVG                                  1761.2 25      AVG         913.4   60    SH                                   1734.2 27      AVG         897.0   59    AVG                                  1631.0 33      AVG         865.1   46    AVG                                  1577.9 41      SH          843.9   60    AVG                                  1545.1 18      SHP         831.4   59    AVG                                  1508.5 43      AVG         800.5   67    AVG                                  1451.6 28      AVG         778.3   59    AVG                                  1434.2 39      SH          732.0   59    AVG                                  1413.9 42      AVG         706.0   65    AVG                                  1384.0 31      AVG         667.4   60    SH                                   1382.1 31      SH          657.8   59    AVG                                  1368.6 34      AVG         618.2   61    SH                                   1346.4 36      AVG         607.6   58    AVG                                  1310.7 36      AVG         507.3   60    AVG                                  1249.0 39      BRD         489.0   59    AVG                                  1235.5 40      BRD         430.0   52    AVG   *                              1221.0 37      AVG                                                            ______________________________________                                         Band Freq.: Band Frequencies in Wavenumbers (CM.sup.-1)                       Inten.: Intensity in Percent Transmittance (% T)                              Date Type in Local Peak Region:                                               BRD = Broad;                                                                  AVG = Average;                                                                SHP = Sharp;                                                                  SH = shoulder                                                                 Peak List Edited.                                                             * Indicates Peaks Added.                                                 

¹³ C-Nuclear Magnetic Resonance (NMR) Spectrum:

The ¹³ C-NMR spectrum of antibiotic U-64,815 is shown in FIG. 3 of thedrawings. The ¹³ C-NMR spectrum was observed on a Varian XL-200Spectrometer (50 mHz) on a solution (ca. 0.5 ml., ca. 40 mg/ml) of thesample of the antibiotic in deutero-methanol. The spectrum wascalibrated against internal tetramethylsilane and frequencies wererecorded in ppm downfield from tetramethylsilane.

Solubilities:

Antibiotic U-64,815 is soluble in methanol acetone, methylene chloride,ethanol and dimethylsulfoxide. It is insoluble in water and hexane.

Antimicrobial Spectrum of Antibiotic U-64,815:

Antibiotic U-64,815 is active against various Gram-positive bacteria andH. influenzae as shown in the following table.

Assay:

The antibacterial assay is a standard disc plate assay. Ten 6.35 mmabsorbent discs are saturated with a 1000 μg/ml solution prepared inmethanol (solvent) and air-dried prior to spotting on organism-seededagar plates. The results are as follows:

    ______________________________________                                                              Zone Size (mm)                                          ______________________________________                                        Haemophilus influenzae                                                                          UC 6482   10                                                (Ampicillin-susceptible)                                                      Haemophilus influenzae                                                                          UC 6483    9                                                (Ampicillin-resistant)                                                        Streptococcus pyogenes                                                                          UC 152    13                                                Streptococcus pneumoniae                                                                        UC 41     16 - outer ring                                                               10 - inner ring                                   Staphylococcus aureus                                                                           UC 6685    8                                                (Multiple antibiotic-resistant)                                               Staphylococcus aureus                                                                           UC 6690    8 - light                                        (Multiple antibiotic-resistant)                                               N. gonorrhoeae    UC 3065   16                                                ______________________________________                                    

"UC" is a registered trademark of The Upjohn Company Culture Collection.These cultures can be obtained from The Upjohn Company in Kalamazoo,Mich., upon request.

THE MICROORGANISM

The microorganism used for the production of antibiotic U-64,815 is abiologically pure culture of Streptomyces microspinus, NRRL 12524.

A subculture of this microorganism can be obtained from the permanentcollection of the Northern Regional Research Laboratory, U.S. Departmentof Agriculture, Peoria, Ill., U.S.A. Its accession number in thisdepository is NRRL 12524. It should be understood that the availabilityof the culture does not constitute a license to practice the subjectinvention in derogation of patent rights granted with the subjectinstrument by governmental action.

The microorganism of this invention was studied and characterized byAlma Dietz of The Upjohn Research Laboratories.

Streptomyces microspinus, Dietz sp. nov. ["microspinus" denotes thesmall spines found on the surface of the spores], appeared most similaron Ektachrome (Table 1) to Streptomyces impexus NRRL 3809, which isdescribed in Goldstein and Prokop [Goldstein, A. W. and J. F. Prokop.1972. Antibiotic effective against gram-positive bacteria and method ofpreparation. U.S. Pat. No. 3,651,217], and Streptomyes fungicidicusATCC®21013, which is described in Shibata et al [Shibata, M., E.Higashide, K. Hatano, K. Mizuno, M. Asai, and M. Muroi. 1974. Antibioticenduracidin and process for the production thereof. U.S. Pat. No.3,786,142]. All the cultures have open spiral chains of spores withspiny surfaces. S. impexus and S. fungicidicus have abundant chains offlexuous spore chains as well as open-spiral spore chains. The spines onspores of these cultures are more readily apparent than those on sporesof Streptomyces microspinus. All cultures are melanin-negative. All havegray aerial growth when the surface color is matched with the NBS colorchips [SRM 2106. ISCC-NBS Centroid Color Charts. Office of StandardReference Material, Room B311, Chem. Building, National Bureau ofStandards, Washington, D.C. 20234], but only Streptomyces microspinusbelongs to the Gray series of Tresner and Backus [Tresner, H. D., and E.J. Backus. 1963. System of color wheels for streptomycete taxonomy.Appl. Microbiol. 11:335-338]. Streptomyces microspinus is furtherdistinguished from S. impexus and S. fungicidicus by its growth oncarbon compounds (Table 3) in the synthetic medium of Shirling andGottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods forcharacterization of Streptomyces species. Int. J. Syst. Bacteriol.16:313-340] and by its general cultural characteristics (Table 4). Thenew culture grows well in the temperature range of 18°-55° C.; the othercultures do not grow at 45°-55° C. The new culture produces theantibiotic U-64815; S. impexus produces M-411 and S. fungicidicusproduces Enduracidin. It can also be differentiated from cultures in theGray spiral, melanin-negative, spiny spore group in Bergey's Manual, 8thed. [Pridham, T. G., and H. D. Tresner. 1974. Part 17. Actinomycetes andrelated organisms. Family VII. Streptomycetaceae Waksman and Henrici1943. Genus I. Streptomyces Waksman and Henrici 1943. Table 17.42h ofthe Gray series. Pages 782-785 in Buchanan and Gibbons, eds., Bergey'sManual of Determinative Bacteriology, 8th ed. The Williams and WilkinsCo., Baltimore]. Therefore, it is proposed that Streptomyces microspinusbe considered a new species of the actinomycete genus Streptomyces. Itis understood that in accordance with the Rules of Nomenclature ofBacteria [Lapage, S. P., P. H. A. Sneath, E. F. Lessel, V. B. D.Skerman, H. P. R. Seeliger, and W. A. Clark, ed. 1975. Internationalcode of nomenclature of bacteria, 1976 Revision. American Society forMicrobiology, Washington, D.C.], this is the type strain and that,should another strain be found, the type strain would also be the typesubspecies.

Streptomyces microspinus Dietz sp. nov. NRRL 12524.

Color Characteristics: Aerial mycelium prednominantly gray.Melanin-negative. The color pattern of the culture on Ektachrome isgiven in Table 1. Reference color characteristics are given in Table 2.The culture may be placed in the Gray (GY) color series of Tresner andBackus [Tresner, H. D., and E. J. Backus, Ibid.].

Microscopic Characteristics: Spore chains are predominantly open-spiral.Spores are oval, 1.1×0.76 μm. Many spores have depressions like those ofred blood cells; others have a double collapsed appearance which gives aridged appearance. The surface of the spores is covered with bluntspines. Spore surface detail is best determined at magnifications of10,000 to 20,000 with the scanning electron microscope. Fourteen daycover glass preparations were used for the microscopic examinations.

Growth on Carbon Compounds: See Table 3.

Whole Cell Analysis: L-diaminopimelic acid was detected.

Culture Characteristics-General: See Table 4.

Temperature: Growth was good at 18°-55° C. on Czapek's sucrose andMaltose-tryptone agars and at 18°-45° C. on Bennett's agar.

Source: Kansas soil.

Type Strain: Streptomyces microspinus sp. nov. NRRL 12524.

                                      TABLE 1                                     __________________________________________________________________________    Color Characteristics* on Ektachrome**                                               Deter-                                                                            S. microspinus                                                                          S. impexus                                                                             S. fungicidicus                                        mina-                                                                             NRRL 12524                                                                              NRRL 3809                                                                              ATCC 21013                                      Agar Medium                                                                          tion                                                                              Chip                                                                             Color  Chip                                                                             Color Chip                                                                             Color                                        __________________________________________________________________________    Bennett's                                                                            S   233                                                                              pale gray                                                                            32 grayish                                                                             70 light                                                                yellowish                                                                              orange                                                               pink     yellow                                              R   72 dark orange                                                                          70 light 70 light                                                      yellow    orange   orange                                                               yellow   yellow                                       Czapek's                                                                             S   233                                                                              pale gray                                                                            32 grayish                                                                              9 pink white                                   sucrose                 yellowish                                                                     pink                                                         R   32 grayish                                                                              32 grayish                                                                              9 pink white                                                 Yellowish yellowish                                                           pink      pink                                                  Maltose-                                                                             S   233                                                                              pale gray                                                                            31 pale yellow                                                                          7 pale pink                                    tryptone                pink                                                         R   72 dark orange                                                                          67 brilliant                                                                           68 strong                                                     yellow    orange   orange                                                               yellow   yellow                                       Peptone-                                                                             S   71 moderate                                                                             32 grayish                                                                             71 moderate                                     iron          orange    yellowish                                                                              orange                                                     yellow    pink     yellow                                              R   68 strong 68 strong                                                                              68 strong                                                     orange    orange   orange                                                     yellow    yellow   yellow                                       0.1%   S   32 grayish                                                                              32 grayish                                                                              7 pale pink                                    Tyrosine      yellowish yellowish                                                           pink      pink                                                         R   38 dark   38 dark  38 dark                                                       reddish   reddish  reddish                                                    orange    orange   orange                                       Casein S   233                                                                              pale gray                                                                            32 grayish                                                                             10 pinkish                                      starch                  yellowish                                                                              gray                                                                 pink                                                         R   93 yellowish                                                                            76 light 93 yellowish                                                  gray      yellowish                                                                              gray                                                                 brown                                                 __________________________________________________________________________     S = Surface                                                                   R = Reverse                                                                   *Growth on media in tubes was photographed after seven days incubation at     28° C. Color was determined by comparison with NBS color chips [SP     440. Color: Universal Language and Dictionary of Name. U.S. Government        Printing Office, Washington, D.C. 20402; SRM 2106. Ibid.].                     **Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete          classification. Ann. N.Y. Acad. Sci. 60:152-154 and Dietz, A. and D. W.       Thayer (ed.). 1980. Actinomycete Taxonomy (Procedures for Studying Aerobi     Actinomycetes with Emphasis on the Streptomycetes). SIM Special               Publication Number 6. Soc. for Ind. Microbiol. Arlington, VA.            

                                      TABLE 2                                     __________________________________________________________________________    Reference Color Characteristics*                                                     Deter-                                                                            S. microspinus                                                                         S. impexus                                                                              S. fungicidius                                         mina-                                                                             NRRL 12524                                                                             NRRL 3809 ATCC 21013                                      Agar Medium                                                                          tion                                                                              Chip                                                                             Color Chip                                                                             Color  Chip                                                                             Color                                        __________________________________________________________________________    Bennett's                                                                            S   266                                                                              dark gray                                                                           90 grayish                                                                              90 grayish                                                             yellow    yellow                                              R   58 moderate                                                                            90 grayish                                                                              70 light orange                                               brown    yellow    yellow                                              P   71 moderate                                                                            --   --   --   --                                                       orange                                                                        yellow                                                          Czapek's                                                                             S   45 light gray-                                                                         63 light  63 light brown-                                 sucrose       ish red- brownish  ish gray                                                   dish brown                                                                             gray                                                          R   93 yellowish                                                                           73 pale orange                                                                          73 pale orange                                                gray     yellow    yellow                                              P   --   --  --   --   --   --                                         Maltose-                                                                             S   266                                                                              dark gray                                                                           93 yellowish                                                                            93 yellowish                                    tryptone               gray      gray                                                R   57 light brown                                                                         71 moderate                                                                             71 moderate                                                            orange    orange                                                              yellow    yellow                                              P   87 moderate                                                                            87 moderate                                                                             87 moderate                                                   yellow   yellow    yellow                                       Yeast  S   23 dark red-                                                                           22 reddish                                                                              63 light brown-                                 extract-      dish gray                                                                              gray      ish gray                                     malt   R   57 light brown                                                                         71 moderate                                                                             71 moderate                                     extract                orange    orange                                       (ISP-2)                yellow    yellow                                              P   71 moderate                                                                            87 moderate                                                                             87 moderate                                                   orange   yellow    yellow                                                     yellow                                                          Oatmeal                                                                              S   45 light gray-                                                                         63 light brown-                                                                         63 light brown-                                 (ISP-3)       ish red- ish gray  ish gray                                                   dish brown                                                             R   29 moderate                                                                            71 moderate                                                                             71 moderate                                                   yellowish                                                                              orange    orange                                                     pink     yellow    yellow                                              P   30 dark yel-                                                                           87 moderate                                                                             87 moderate                                                   lowish pink                                                                            yellow    yellow                                       Inorganic                                                                            S   23 dark red-                                                                           63 light brown-                                                                         22 reddish                                      Salts         dish gray                                                                              ish gray  gray                                         Starch R   73 pale orange                                                                         71 moderate                                                                             71 moderate                                     (ISP-4)       yellow   yellow    yellow                                              P   79 light gray-                                                                         80 grayish                                                                              80 grayish                                                    ish yel- yellowish yellowish                                                  lowish   brown     brown                                                      brown                                                           Glycerol-                                                                            S   266                                                                              dark gray                                                                           93 yellow gray                                                                          93 yellow gray                                  asparagine                                                                           R   57 light brown                                                                         71 moderate                                                                             71 moderate                                     (ISP-5)                orange    orange                                                              yellow    yellow                                              P   --   --  87 moderate                                                                             87 moderate                                                            yellow    yellow                                       __________________________________________________________________________     S = Surface                                                                   R = Reverse                                                                   P = Pigment                                                                   *Color determination was made on growth on plates incubated 14 days at        28° C. Color was determined by comparison with NBS color chips [SP     440, Ibid. and SRM 2106, Ibid.                                           

                  TABLE 3                                                         ______________________________________                                        Growth on Carbon Compounds in the Synthetic                                   Medium of Shirling and Gottlieb*                                              Synthetic Medium                                                                         S. microspinus                                                                           S. impexus S. fungicidicus                              (ISP-9)    NRRL 12524 NRRL 3089  ATCC 21013                                   ______________________________________                                        Negative Control                                                                         -          -          -                                            (No carbon cpd.)                                                              Positive Control                                                                         +          ++         +                                            (D-glucose)                                                                   L-arabinose                                                                              ++         ++         ++                                           Sucrose    -          ±       ±                                         D-xylose   ++         ++         ++                                           Inositol   ++         ++         ++                                           D-mannitol ++         ++         ++                                           D-fructose +          ++         ++                                           Rhamnose   ++         ++         ++                                           Raffinose  -          -          -                                            Cellulose  -          -          -                                            ______________________________________                                         ++ = Strong utilization                                                       + = Positive utilization                                                      ± = Doubtful utilization                                                   - = No utilization                                                            *Shirling, E. B. et al., Ibid.                                           

                                      TABLE 4                                     __________________________________________________________________________    Culture Characteristics - General                                                    Deter-                                                                        mina-                                                                             S. microspinus                                                                        S. impexus                                                                            S. fungicidicus                                    Agar Medium                                                                          tion                                                                              NRRL 12524                                                                            NRRL 3809                                                                             ATCC 21013                                         __________________________________________________________________________    Agar                                                                          Peptone-                                                                             S   pale gray                                                                             white center;                                                                         gray center;                                       iron               pink-tan edge                                                                         white edge                                                R   tan     light yellow-                                                                         yellow                                                                tan                                                               P     --      --      --                                                      O   melanin melanin melanin                                                       negative                                                                              negative                                                                              negative                                           Calcium                                                                              S   gray    gray    gray                                               maleate                                                                              R   pale gray                                                                             gray    gray-yellow                                               P     --      --      --                                                      O   malate not                                                                            malate not                                                                            malate not                                                    solubilized                                                                           solubilized                                                                           solubilized                                        Glucose-                                                                             S   gray-pink                                                                             yellow vegeta-                                                                        cream-gray                                         asparagine         growth                                                            R   pink-tan                                                                              deep yellow                                                                           deep yellow                                               P   pale pink                                                                               --      --                                               Skim milk                                                                            S   brown vegeta-                                                                         gray center;                                                                          trace gray                                                    tive growth                                                                           cream edge                                                        R   tan     orange  orange                                                    P   tan     orange  orange                                                    O   casein not                                                                            casein solu-                                                                          casein solu-                                                  solubilized                                                                           bilized bilized                                            Tyrosine                                                                             S   pale gray                                                                             gray center;                                                                          cream center;                                                         cream edge                                                                            gray-white                                                                    edge                                                      R   orange-tan                                                                            orange-yellow                                                                         orange-tan                                                P   orange-tan                                                                            pale orange                                                                           orange-tan                                                O   tyrosine                                                                              tyrosine                                                                              tyrosine                                                      solubilized                                                                           solubilized                                                                           solubilized                                        Xanthine                                                                             S   pale gray on                                                                          gray cream                                                                            cream                                                         edge; tan                                                                     vegetative                                                                    center                                                                    R   yellow-cream                                                                          orange  cream                                                     P   pale yellow                                                                             --    pale yellow                                               O   xanthine                                                                              xanthine not                                                                          xanthine not                                                  solubilized                                                                           solubilized                                                                           solubilized                                        Nutrient                                                                             S   blue-gray                                                                             gray center;                                                                          cream                                              starch             cream edge                                                        R   yellow cream                                                                          yellow  yellow                                                    P   pale yellow                                                                           pale yellow                                                                           pale yellow                                               O   starch solu-                                                                          starch par-                                                                           starch par-                                                   bilized tially solu-                                                                          tially solu-                                                          bilized bilized                                            Yeast  S   lavender-gray                                                                         gray-cream                                                                            gray with cream                                    extract-                   edge                                               malt   R   orange-tan                                                                            yellow-orange                                                                         yellow                                             extract            tan                                                               P   pale orange-                                                                          pale yellow                                                                           pale yellow                                                   tan                                                                Peptone-                                                                             S   light gray-                                                                           light gray-                                                                           colorless vegeta-                                  yeast      pink    white   tive with very                                     extract-                   slight trace                                       iron                       gray-white                                         (ISP-6)                                                                              R   yellow-tan                                                                            yellow-tan                                                                            yellow-tan                                                P   yellow-tan                                                                            yellow-tan                                                                            yellow-tan                                                O   melanin melanin melanin                                                       negative                                                                              negative                                                                              negative                                           Tyrosine                                                                             S   steel gray                                                                            mottled gray-                                                                         light steel gray                                   (ISP-7)            yellow with                                                                   yellow exudate                                                    R   light gray                                                                            yellow-tan                                                                            light yellow-gray                                         P     --    trace yellow-                                                                           --                                                                  tan                                                               O   melanin melanin melanin                                                       negative                                                                              negative                                                                              negative                                           Broth                                                                         Synthetic                                                                            S   trace white                                                                           trace white                                                                           slight white                                       nitrate    aerial on                                                                             aerial on                                                                             aerial on                                                     surface surface surface                                                       pellicle                                                                              pellicle                                                                              pellicle                                                  P     --      --      --                                                      O   fine growth                                                                           trace growth                                                                          slight growth                                                 throughout                                                                            throughout                                                                            throughout                                                    flocculent                                                                            flocculent                                                                            flocculent                                                    growth at base                                                                        growth at base                                                                        growth at base                                                growth color-                                                                         growth color-                                                                         growth color-                                                 less    less    less                                                          nitrates not                                                                          nitrates                                                                              nitrates                                                      reduced reduced reduced                                            Nutrient                                                                             S   gray white                                                                            white aerial                                                                          white aerial                                       nitrate    aerial on                                                                             on surface                                                                            on surface                                                    surface pellicle                                                                              pellicle                                                      pellicle                                                                  P     --      --      --                                                      O   flocculent                                                                            flocculent                                                                            flocculent                                                    yellowish                                                                             yellowish                                                                             yellowish                                                     bottom growth                                                                         bottom growth                                                                         bottom growth                                                 nitrates not                                                                          nitrates                                                                              nitrates                                                      reduced reduced reduced                                            Litmus S   trace gray-                                                                           pale gray-                                                                            pale gray-                                         milk       white aerial                                                                          white aerial                                                                          white aerial                                                  on blue-                                                                              on surface                                                                            on surface                                                    gray surface                                                                          pellicle                                                                              pellicle                                                      ring    blue-gray-tan                                                                         blue-gray-tan                                                         surface ring                                                                          surface ring                                              P     --      --      --                                                      O   litmus reduced                                                                        litmus reduced                                                                        litmus reduced                                                pH 7.7  pH 7.6  pH 7.4                                             Gelatin                                                                       Plain  S   trace tan                                                                             trace tan                                                                             trace tan                                                     vegetative                                                                            vegetative                                                                            vegetative                                                    growth at                                                                             growth at                                                                             growth at                                                     surface surface surface                                                   P   trace olive                                                                           trace yellow                                                                          trace yellow                                              O   liquefaction                                                                          liquefaction                                                                          liquefaction                                                  complete                                                                              complete                                                                              complete                                           Nutrient                                                                             S   tan vegetative                                                                        tan vegetative                                                                        tan vegetative                                                growth at                                                                             growth at                                                                             growth at                                                     surface surface surface                                                   P     --    tan     yellow                                                    O   liquefaction                                                                          liquefaction                                                                          liquefaction                                                  complete                                                                              complete                                                                              complete                                           __________________________________________________________________________     S = Surface (aerial growth unless otherwise noted)                            R = Reverse                                                                   P = Pigment                                                                   O = Other Characteristics                                                

The compound of the invention process is produced when the elaboratingorganism is grown in an aqueous nutrient medium under submerged aerobicconditions. It is to be understood, also, that for the preparation oflimited amounts surface cultures and bottles can be employed. Theorganism is grown in a nutrient medium containing a carbon source, forexample, an assimilable nitrogen compound or proteinaceous material.Preferred carbon sources include glucose, brown sugar, sucrose,glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like.Preferred nitrogen sources include cornsteep liquor, yeast, autolyzedbrewer's yeast with milk solids, soybean meal, cottonseed meal,cornmeal, milk solids, pancreatic digest of casein, fish meal,distiller's solids, animal peptone liquors, meat and bone scraps, andthe like. Combinations of these carbon and nitrogen sources can be usedadvantageously. Trace metals, for example, zinc, magnesium, manganese,cobalt, iron, and the like, need not be added to the fermentation mediumsince tap water and unpurified ingredients are used as components of themedium prior to sterilization of the medium.

Production of the compound by the invention process can be effected atany temperature conducive to satisfactory growth of the microorganism,for example, between about 18° and 40° C., and preferably between about20° and 28° C. Ordinarily, optimum production of the compound isobtained in about 3 to 15 days. The medium normally remains alkalineduring the fermentation. The final pH is dependent, in part, on thebuffers present, if any, and in part on the initial pH of the culturemedium.

When growth is carried out in large vessels and tanks, it is preferableto use the vegetative form, rather than the spore form, of themicroorganism for inoculation to avoid a pronounced lag in theproduction of the compound and the attendant inefficient utilization ofthe equipment. Accordingly, it is desirable to produce a vegetativeinoculum in a nutrient broth culture by inoculating this broth culturewith an aliquot from a soil, liquid N₂ agar plug, or a slant culture.When a young, active vegetative inoculum has thus been secured, it istransferred aseptically to large vessels or tanks. The medium in whichthe vegetative inoculum is produced can be the same as, or differentfrom, that utilized for the production of the compound, so long as agood growth of the microorganism is obtained.

A variety of procedures can be employed in the isolation andpurification of the compound produced by the subject invention fromfermentation beers. Isolation can be accomplished by extraction withsolvents such as methylene chloride, acetone, butanol, ethyl acetate andthe like; and silica gel chromatography can be used to purify crudepreparations of the antibiotic.

In a preferred recovery process, the compound produced by the subjectprocess is recovered from the culture medium by separation of themycelia and undissolved solids by conventional means, such as byfiltration or centrifugation and solvent extraction of the filteredbroth. The filtrate can be extracted with a solvent for antibioticU-64,815, for example, methylene chloride, and the extract evaporatedunder reduced pressure to an aqueous concentrate. This preparation canbe purified by chromatography on silica gel. The solvent system used forthe chromatography is methylene chloride:methanol (95:5) (v/v) and(90:10). Antibiotic U-64,185 is shown at R_(f) =0.50-0.55 silica gel Gplates in MeCl₂ /MeOH 9:1 using standard bioautography on Bacillussubtilis.

The antibiotic of the subject invention also can be recovered from thefermentation broth by resin sorption on a resin comprising a non-ionicmacro porous copolymer of styrene cross linked with divinylbenzene.Suitable resins are Amberlite XAD-2; XAD-4, and XAD-7, according to theprocedure disclosed in U.S. Pat. No. 3,515,717. (Amberlite resins areavailable from Rohm and Haas, Philadelphia, PA). The antibiotic can beeluted from said resins by using acetone.

Salts of antibiotic U-64,815 also can be formed with inorganic ororganic bases. Such salts can be prepared, as for example, by suspendingantibiotic U-64,815 in water, adding a dilute base until the pH of thesuspension is about 10.0 to 11.0, and freeze-drying to provide a driedresidue consisting of the U-64,815 salt. Antibiotic U-64,815 salts withinorganic cations which can be formed include the sodium, potassium, andcalcium salts. Other salts of U-64,815, including those with inorganicbases such as primary, secondary, and tertiary monoamines as well aswith polyamines, also can be formed using the above-described or othercommonly employed procedures. Other valuable salts are obtained withtherapeutically effective bases which impart additional therapeuticeffects thereto. Such bases are, for example the purine bases such astheophyllin, theobromine, caffeine, or derivatives of such purine bases;antihistaminic bases which are capable of forming salts with weak acids,pyridine compounds such as nicotinic acid amide, isonicotinic acidhydrazide, and the like; phenylalkylamines such as adrenaline,ephedrine, and the like; choline, and others. Salts of U-64,815 can beused for the same biological purposes as the parent compound.

The following examples are illustrative of the process and product ofthe invention, but are not to be construed as limiting. All percentagesare by weight and all solvent mixture proportions are by volume unlessotherwise noted.

EXAMPLE 1 A. Fermentation

A biologically pure culture of Streptomyces microspinus, NRRL 12524, isused to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml ofsterile medium consisting of the following ingredients:

    ______________________________________                                                        g/liter                                                       ______________________________________                                        Cerelose          25                                                          Pharmamedia*      25                                                          Deionized water q.s.                                                                            1 liter                                                     ______________________________________                                         *Pharmamedia is an industrial grade of cottonseed flour produced by           Traders Oil Co, Fort Worth, TX.                                          

The seed medium post-sterilization pH is ˜6.5. The seed inoculum isgrown for three days at 28° C. on a Gump rotary shaker operating at 250rpm and having a 21/2 inch stroke.

Seed inoculum (100 ml), prepared as described above, is used toinoculate 500-ml fermentation flasks (Erlenmeyer) containing 100 ml ofsterile fermentation medium consisting of the following ingredients:

    ______________________________________                                                        g/liter                                                       ______________________________________                                        Cerelose          10                                                          Bacto-Peptone (Difco)                                                                            5                                                          Black Strap Molasses*                                                                           20                                                          Tap Water         Balance                                                     ______________________________________                                         *Supplied by Knappen Milling Co., Augusta, MI.                           

The pH is adjusted to 7.2 with NaOH before autoclaving.

The inoculated fermentation medium is incubated at a temperature of 28°C. for 5 days on a Gump rotary shaker operating at 250 rpm and having a21/2 inch stroke.

A typical five-day fermentation has the following titers of antibioticin the fermentation broth:

    ______________________________________                                               Day  Assay, BU/ml                                                      ______________________________________                                               2    8.0                                                                      3    >16                                                                      4    >16                                                                      5    >16                                                               ______________________________________                                    

The assay is a Bacillus subtilis disc plate assay using 0.1 M potassiumphosphate buffer, pH 5.0 as diluent.

B. Recovery

Whole beer, from a fermentation as described above, is filtered andadjusted to neutral pH with HCl and filtered with 5% diatomaceous earth(wt/v). The clear beer is then defatted with two half volumes ofSkellysolve B (isomeric hexanes). The defatted beer is then extractedwith two half volumes of methylene chloride and these extracts arecombined and reduced in volume.

    ______________________________________                                        1-liter whole beer adj. to pH 7 with HCl                                       ##STR1##                                                                     Clear Beer                                                                    (1/2" dipped disc bioassay)                                                                    Whole Beer Clear Beer                                        Organism         Zone (mm)  Zone (mm)                                         ______________________________________                                        B. subtilis      29         29                                                M. luteus        25         24                                                C. perfringens   28         28                                                ______________________________________                                                 1st     2nd       Combined                                           Organism Extract Extract   Extracts                                                                              Spent Beer                                 ______________________________________                                        Skellysolve B                                                                 B. subtilis                                                                            0       0         0       28                                         M. luteus                                                                              0       0         0       23                                         C. perfringens                                                                         0       0         0       27                                         Me.sub.2 Cl.sub.2                                                             B. subtilis                                                                            27      19        27      21                                         S. lutea 23       0        21      16                                         C. perfringens                                                                         27      18        27      20                                         ______________________________________                                    

C. Purification

A 21.5 g sample of a CH₂ Cl₂ extract, prepared as described above, isdissolved in a minimal volume of CH₂ Cl₂ and passed onto the head of a5' column packed with 1400 g of Merck Silica Gel 60 slurried in CH₂ Cl₂.A flow rate of ca. 7 ml/min is established with a solvent of CH₂ Cl₂/MeOH; 90/5; 20 ml fractions are collected. After 8.7 liters, thesolvent is changed to CH₂ Cl₂ /MeOH; 90/10.

Fractions are analyzed by thin layer chromatography (TLC) using AnaltechSilica gel GF plates run in CH₂ Cl₂ /MeOH, 9/1. The component ofinterest is first detected by quenching under U.V. 254 nm light followedby spraying with a p-anisaldehyde reagent (EtOH:H₂ SO₄ :AceticAcid:p-anisaldehyde, 90:5:1:5) and heating. U-64,815 runs in this systemwith R_(f) =0.50-0.55 and develops a deep purple coloration whenprocessed with this reagent.

Fractions 436-570 are pooled to give 4.25 g of a brown solid which has˜32 bu/mg when assayed with 1/2" discs on B. subtilis by a dilutionbioassay. (One bu is defined as the amount of antibiotic needed toproduce a 20 mm zone).

The 4.25 g solid is dissolved in a minimal volume of MeOH/H₂ O, 80/20and loaded onto the head of a 25 mm I.D. column packed with 42.5 g ofWhatman LRP-1 reverse phase equilibrated with the solvent. A flow rateof 1.5 ml/min is established with MeOH/H₂ O, 80/20 while 50 ml fractionsare collected. After the seventh fraction, the solvent is changed to100% MeOH. Fractions are analyzed in the identical manner as before andalso with dipped discs plated against B. subtilis. The active fractionscontaining the best material as seen by TLC are pooled and reduced involume to give 2.75 g of an off-white powder of essentially pureantibiotic U-64,815.

We claim:
 1. A process for preparing antibiotic U-64,815, which in itsessentially pure form has the following characteristics:(a) molecularweight range of 1,104-1,324; (b) molecular formula: C₆₀₋₆₅ H₈₂₋₁₀₂ N₁₋₃O₁₈₋₂₅ ; (c) is insoluble in water and hexane, soluble in methanol,acetone, methylene chloride, ethanol, and dimethylsulfoxide; (d) acharacteristic ¹³ C-NMR spectrum as shown in FIG. 3 of the drawings; (e)a characteristic UV spectrum as shown in FIG. 2 of the drawings; and,(f) a characteristic infrared absorption spectrum when dissolved in amineral oil mull as shown in FIG. 1 of the drawings;which comprisescultivating Streptomyces microspinus, having the identifyingcharacteristics of NRRL 12524, in an aqueous nutrient medium underaerobic conditions until substantial antibiotic U-64,815 activity isimparted to said medium.
 2. A process, according to claim 1, whereinsaid aqueous nutrient medium contains a source of assimilablecarbohydrate and assimilable nitrogen.